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Page 613 out of 618

Correction of Murine Hemophilia a by Hematopoietic Stem Cell Gene Therapy

Moayeri M, Hawley TS, Hawley RG
Product: Tetanus Toxoid from Clostridium tetani

FVIII and Tetanus Toxoid Immunizations:

... Two of the 800 cGy-irradiated mice (mice #26 and #27) and four naive E16 mice were immunized with tetanus toxoid (List Biological Laboratories, Inc., Campbell, CA, USA) 3 weeks after the final FVIII injection as follows: 4 μg tetanus toxoid was mixed with 1 mg Al (OH)3adjuvant (Alhydrogel 2%; Accurate Chemical & Scientific Corporation, Westbury, NY, USA), diluted in sterile saline to a total volume of 108 μl and injected intraperitoneally. Two weeks later, sera from these mice was assessed for the presence of anti-tetanus toxoid antibodies by ELISA (described below).

PubMed ID: 16226058

Anthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs the Adaptive Immune Response

Alileche A, Serfass ER, Muehlbauer SM, Porcelli SA, Brojatsch J
Product: Anthrax Lethal Factor (LF),Recombinant from B. anthracis

Materials and Methods:

... Recombinant anthrax LF was obtained from List Biological Laboratories (Campbell, California, United States). PA was generously provided by Dr. Steven Leppla (National Institutes of Health). PA and LF were reconstituted in water at 500 ng/ml and 250 ng/ml, respectively [35]. Recombinant LF and PA were produced in B. anthracis and were free of LPS contamination as indicated by the manufacturer, and by a lack of CD86 up-regulation on immature cells, as measured by flow cytometry.

Cell death and viability assays:

Cell viability was measured by analysis of MTT cleavage to formazan by succinate dehydrogenases in living cells [37]. For the colorimetric MTT assay, cells were exposed to LT (500 ng/ml PA and 250 ng/ml LF), and the MTT solution (5 mg/ml MTT in PBS) was added directly to wells and incubated at 37 °C for 4 h. The dye was solubilized with acidic isopropanol (25 mM HCl and 0.5% SDS in isopropanol), and the absorbance was measured at 570 nm. ...

Western blotting:

Cells were cultured in 24-well plates and treated with LT.  ...

In vivo assay:

Ten-week-old female BALB/c mice were injected intraperitoneally with LT [3,4]. Mice were sacrificed 0, 3, 6, and 24 h after LT injection. Spleens were harvested and treated with 400 U/ml collagenase D (Roche) to release DCs. Levels of splenic DCs (CD11c+, MHC class II+), macrophages (CD11b+, MHC class II+), and circulating B cells (B220+) and T cells (CD3+) were determined by flow cytometry using fluorescently labeled antibodies to surface markers.

 

LT (Lethal Toxin) = Lethal Factor (LF) + Protective Antigen (PA)

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

PubMed ID: 16254597

Characteristics of Physical Characteristics of rLF and rPA: Effects on Enzymatic : Effects on Enzymatic Activity and Binding

Shine, N., Le, C., Eaton, L., Crawford, K.
Product: Unquenched Calibration Peptide for MAPKKide® 530 for Anthrax Lethal Factor

Materials:

Recombinant lethal factor (Prod #172), protective antigen (Prod #171), MAPKKideTM (Prod #530), and MAPKKideTM Unquenched Calibration Peptide (Prod #539) are products of List Biological Laboratories.

Methods - The above materials were used in the following (refer to poster for details):

  • HPLC
  • Enzymatic activity
  • Continuous fluorimetric assays
  • LF-PA63 binding assay
  • LF enzymatic activity as a function of ZnCl2

A Soluble Receptor Decoy Protects Rats against Anthrax Lethal Toxin Challenge

Scobie HM, Thomas D, Marlett JM, Destito G, Wigelsworth DJ, Collier RJ, Young JA, Manchester M
Product: Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Materials and methods:

Rat LeTx challenge experiments were performed in accordance with protocols approved by the Scripps Institutional Animal Care and Use Committee. Male Fisher 344 rats (180–200g; Harlan) were anesthetized with isofluoranes and were inoculated with LeTx mixture through a jugular-vein cannula. LeTx was prepared with 40 mg of PA and 8 mg of LF per rat (List Biological Laboratories). For rats that received sCMG2 and LeTx, sCMG2 was added to the LeTx mixture and was coinjected in a 500-mL volume. Rats recovered from anesthesia within 5 min and were monitored for symptoms of intoxication and death. Statistical analysis was conducted on the basis of Student’s unpaired t test (Prism, version 4; GraphPad). For rats that died overnight, statistical analysis was based on the last observed postinoculation time point.

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

PubMed ID: 16107958

Efficient synthetic inhibitors of anthrax lethal factor

Forino M, Johnson S, Wong TY, Rozanov DV, Savinov AY, Li W, Fattorusso R, Becattini B, Orry AJ, Jung D, Abagyan RA, Smith JW, Alibek K, Liddington RC, Strongin AY, Pellecchia M
Product: MAPKKide® Peptide Substrate (o-Abz/Dnp)for Anthrax Lethal Factor

MATERIALS AND METHODS:

... Recombinant LF and MAPKKide were both purchased from List Biological Laboratories (Campbell, CA). ...

Fluorescence Peptide Cleavage Assay:

Cleavage reactions (100 μl each) were performed in a 96-well plate. Each reaction contained MAPKKide (4 μM) and LF (50 nM) in 20 mM Hepes, pH 7.4, and the small-molecule inhibitor. Kinetics of the peptide cleavage was examined for 30 min by using a fluorescent plate reader at excitation and emission wavelength at 485 and 590 nm, respectively.

The Km and Vmax values of the MAPKKide cleavage by LF were determined at 25°C by using the same experimental condition described above for the fluorescence screening assay but with increasing MAPKKide concentrations (2, 3, 5, 8, and 10 μM). The Ki and Km(app) were calculated at a fixed 10 μM inhibitor concentration. All constant values were definitely evaluated by fitting the data to the Lineweaver–Burk plot.

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

PubMed ID: 15983377